The present invention relates to a method and apparatus for the introduction of selected chemical elements present in solid or liquid samples into an accelerator mass spectrometry (AMS) system or other analytical instrument.
AMS is a powerful tool for the ultra-sensitive detection of 14C and 3H in biological samples, with proven applicability to current problems in environmental toxicology and human carcinogenesis. For 14C detection, AMS has 1000-fold higher sensitivity than liquid scintillation decay counting, thereby allowing the quantization of attomole (10xe2x88x9218 mole), or smaller, samples. Almost all existing radiocarbon AMS systems require that the sample to be analyzed be introduced into the ion source as solid graphite. Graphitization is a lengthy process (typically taking 6-10 hours) and considerable skill is required to produce layers of uniform composition and thickness and to prevent sample contamination. For example see the paper by J. S. Vogel, K. W. Turteltaub, J. S. Felton, B. L. Gledhill, D. E. Nelson, J. R. Southon, I. D. Proctor and J. C. Davis Nucl. Instr. and Meth. B52 (1990) 524, incorporated herein by reference. Therefore, the analysis of biological samples by AMS requires highly specialized sample preparation procedures that are not compatible with standard chromatographs. This requirement has been a major impediment to the use of AMS in the biomedical sciences.
Liquid chromatography is the technique of choice for high performance separation of large, non-volatile or polar molecules such as proteins, carbohydrates, peptides, and oligonucleotides. The coupling of a liquid chromatograph (LC) to an AMS is particularly challenging because the interface must provide for the efficient conversion of biological molecules in a variety of solvents into CO2 or H2, and must do so with high sample transfer efficiency, good peak shape retention, and minimal contamination with naturally occurring 14C or 3H from other sources such as solvents and previous samples.
To prepare a liquid-phase sample for AMS, the interface must efficiently convert the desired isotope into one or more gaseous compounds suitable for introduction into the ion source. Negative ion sources that allow the sample to be introduced as gaseous CO2 (or H2) have been developed, and have been shown to have sufficiently low sample-to-sample memory for detection of 14C at or near modern abundance. For example, see the paper by C. R. Bronk and R. E. M. Hedges, Nucl. Instr. Meth. Phys. Res. B29(1987) 45; also R. Middleton, J. Klein and D. Fink, Nucl. Instr. Meth. Phys. Res. B43 (1989) 231, incorporated herein by reference. In the ion source, CO2 (or H2) is converted to Cxe2x88x92 (or Hxe2x88x92) for injection into the accelerator mass spectrometer. The AMS ion source may produce positive ions as an intermediate step, as described in U.S. Pat. No. 5,438,194, entitled xe2x80x9cUltra-Sensitive Molecular Identifierxe2x80x9d, by Koudijs et al. A sample chromatogram is illustrated in FIG. 1. However, the applicability of GC-AMS is limited to volatile substances.
For some isotopes, such as 14C, it is important to strip a large fraction of the solvent accompanying the analyte. The extremely low naturally occurring background of tritium lowers the concentration at which samples can be introduced before separation of analyte from sample matrix (e.g., solvent) becomes necessary. The natural abundance of 3H (3H:1H) is xe2x89xa610xe2x88x9215, at least 3 orders-of-magnitude lower than the natural abundance of 14C. The impact of the lower natural abundance of 3H on AMS measurement capabilities can be seen from the following considerations. If it is assumed that the current produced by the AMS ion source is 25 xcexcA, then the particle current of Hxe2x88x92 or Cxe2x88x92 is 1016 ions/min. For 3H detection, a transport efficiency of 50% and a natural abundance of 10xe2x88x9215 yields a corresponding 3H background of 5 cpm at the AMS detector. Detection of 3H with SNR=10 in 1 minute would therefore require 105 cpm 3H from analyte. In this example, the concentration of 3H-labeled analyte is 2.2 pM (105 cpm 3H÷0.5xc3x971016 cpm H, multiplied by 100 moles H/L) and the volumetric flow rate of sample introduced into the ion source is about 1 nL/min. It is clear from these numbers that, even at these very low sample concentrations and flow rates, accurate AMS detection of 3H without solvent removal is possible with negligible contribution from naturally occurring background.
The limits on direct sample introduction for 14C detection are more difficult to define, but are clearly more stringent. For 14C, the natural abundance of 1.4xc3x9710xe2x88x9212 gives a background count rate of 6,600 cpm under the same assumptions used above. Detection of the same number of 14C atoms from analyte (105 cpm) yields a SNR=1.3. In order to obtain the same statistical accuracy of SNR=10, it would be necessary remove solvent to a level of about one part in 103 prior to AMS analysis. Alternately, higher flow rates of sample into the AMS could be used. In this example, about 900 cpm 14C from analyte (in a background of 6,600 cpm from solvent) would yield a SNR=10 due to counting statistics alone, but it would be necessary to eliminate all other noise contributions at the level of better than 1.5%. For these reasons, accurate 14C detection by AMS without solvent removal is extremely difficult.
U.S. Pat. No. 5,438,194, entitled xe2x80x9cUltra-Sensitive Molecular Identifierxe2x80x9d, by Koudijs et al. discloses a system where a liquid or gas chromatograph is coupled directly to the ion source system of an AMS analyzer. However, there are no provisions for desolvation, and the molecular dissociation and ion formation occur in the same process in the ion source itself. In addition, the inventors disclose several designs for the ion source system. However, none of these disclosed designs include a provision for desolvation. At a relatively low solvent flow rate of 1 xcexcL/min, the detection limit for 14C without desolvation will be approximately 0.1 femtomole or higher. This is significantly greater than the target sensitivity for a LC-AMS system of detection of LC peaks containing one attomole (10xe2x88x923 femtomole), or less, of 14C or 3H.
An additional disadvantage of the direct coupling of a liquid chromatograph to the ion source systems described in U.S. Pat. No. 5,438,194 is that molecular dissociation and ion formation (positive or negative) occur in the same process. This coupling of the dissociation and ionization functions will most likely result in a significant dependence of conversion efficiency on input chemical form. The prior art mentions the possibility of dissociating the molecules by high temperature pyrolysis, but there is no detailed description of what compounds are to be formed and whether the dissociation and ionization functions will be separated in this case.
Systems coupling a liquid chromatograph through a conversion reactor to a standard mass spectrometer have been developed for IRMS. These systems include the xe2x80x9cmoving wirexe2x80x9d system as described by R. J. Caini and J. T. Brenna, Anal, Chem. 65 (1993) 3497, and the chemical reaction interface mass spectrometry (CRIMS) interface, as disclosed by M. McLean, M. L. Vestal, Y. Teffera, and F. P. Abramson, J. Chrom. A, 732 (1996), 189. The xe2x80x9cmoving wirexe2x80x9d system has the disadvantage that only a small fraction of the LC eluent can be deposited on the wire, resulting in low analyte transfer efficiency to the IRMS. The CRIMS interface incorporates a Vestec xe2x80x9cUniversal Interfacexe2x80x9d (UI) to separate analyte from solvent. The UI is based on the formation of a highly focused particle beam using thermospray vaporization followed by a multiple-stage desolvation process (momentum separator). The UI operates at normal-bore HPLC flow-rates and uses a high He gas flow to carry the particle beam through the apparatus. The disadvantage of the particle beam desolvation approach for AMS is that existing technology is not scalable to the lower liquid flow rates ( less than 1 xcexcL/min) required for the analysis of extremely small samples.
The recent development of microscale analytical systems is relevant to the development of AMS as a biomedical assay technique. AMS systems, because of their extremely high sensitivity, are uniquely suited to analyze samples introduced using microfluidic devices. Using technology already highly developed in the electronics industry, many university researchers and commercial concerns are producing xe2x80x9clab-on-a-chipxe2x80x9d chemical synthesis and analysis systems that reside on centimeter-sized wafers of silicon, glass, quartz, and polymers. For example, see the paper by R. F. Service, entitled xe2x80x9cLabs on a chip: Coming soon: xe2x80x9cThe pocket DNA sequencerxe2x80x9d, Science 282 (1998) 399; and the paper by M. Freemantle, entitled xe2x80x9cDownsizing chemistryxe2x80x9d, Chemical and Engineering News 77 (1999) 27, both incorporated herein by reference. These systems operate on nanoliter and smaller volume samples and thereby achieve dramatic improvements in sample throughput and speed of analysis while at the same time reducing costs by orders of magnitude.
Matrix-assisted laser desorption/ionization (MALDI) is another commonly used technique in mass spectrometry to desorb and ionize large molecules. In the MALDI technique, the sample is imbedded in a solid matrix, typically organic acid. The analytes are subsequently vaporized and ionized by pulsed laser irradiation, with the goal of retaining the analyte molecular form. This differs from the goal of the present invention, which is to convert selected chemical elements present in the analyte to a common form. A potential disadvantage of MALDI as the initial step of the present invention is the production of background organic molecules from the matrix that may limit the sensitivity achievable with the AMS. Techniques for matrix-free laser desorption using porous silicon as the substrate material have recently been developed that may have advantages over MALDI for AMS applications. For example, see the paper by J. Wei, J. M. Buriak, and G. Siuzdak, Desorption-ionization mass spectrometry on porous silicon. Nature 399 (1999), 243, incorporated herein by reference. Development of on-line LC/MALDI systems is ongoing in research laboratories around the world. Such a system may provide an alternative to electrospray for nebulizing and ionizing large molecules, but does not perform the required conversion of selected chemical elements into the gaseous compounds suitable for introduction into an AMS or other analytical instrument.
Therefore, there is a need for a system and method for converting a non-gaseous sample to a desired gaseous form for analytical processing (e.g., by an AMS), thus allowing standard liquid- and solid phase chemical separation techniques to be utilized to their full potential.
Briefly according to an aspect of the present invention, an interface introduces a solid or liquid sample into an AMS system or other analytical instrument. A non-gaseous sample is introduced into a converter stage, with provisions for separating the analyte molecules from accompanying matrix material (e.g., solvent, organic acid, etc.) if necessary. The converter converts the labeled molecules contained in the sample to one or more standard molecular forms (e.g., CO2, H2, etc.) for introduction into the AMS ion source chamber. The converter stage may be contained within the AMS ion source chamber, or separate from the AMS ion source chamber.
In one class of embodiments of the present invention, a first stage transfers analyte from a solid or liquid form into a stream of carrier gas, with provisions for separating the analyte molecules from accompanying matrix material (e.g., solvent, organic acid, etc.) if necessary. This step is referred to as nebulization for liquid samples, and desorption for solid samples. Alternatively, if it is known that separation of analyte from accompanying matrix material is not required, a liquid sample can be directly injected into the converter using a pipetter, obviating the need for the nebulization step. A second stage converts the labeled molecules contained in the sample to one or more standard molecular forms (e.g., CO2, H2, etc.) for introduction into the AMS ion source chamber.
According to one embodiment of the present invention, a sample is nebulized using electrospray and desired elements in the sample are converted to a predetermined gaseous form, which is input to an AMS system for analysis.
According to a second class of embodiments of the present invention, a sample is deposited onto a solid substrate, and desired elements of the sample are converted to a predetermined gaseous form, which is then provided to an analytical processing device for analysis. There are two general embodiments of this aspect of the invention. In a first embodiment a reaction chamber is connected to an AMS or other apparatus in such a manner as to permit the flow of gases from the one to the other and whose function is to convert the desired elements present in the sample into a gaseous form suitable for introduction into the AMS ion source or other apparatus. Samples to be analyzed are deposited onto or mixed with a solid support that may also be chemically reactive, and placed within the reaction chamber. Chemical conversion is accomplished by specifically directing heat or other forms of energy to the sample or to the substrate where the sample has been applied. If the substrate is itself not reactive, then a reactant gas may be introduced to the reaction chamber during the step of directly heating or applying other forms of energy to the sample or substrate. The application of energy to the sample region serves both to convert the desired elements in the sample to a predetermined chemical form and to release the product chemical from the substrate. A flow of gas passing through the reaction chamber carries the analyte elements in the predetermined chemical form into the AMS ion source.
In a second embodiment, sample is placed on a substrate that is introduced directly into the AMS ion source. The ion source converts the desired elements present in the analyte into ionic species suitable for extraction and injection into the AMS system. Conversion may take place via bombardment with a cesium beam, as in a cesium sputter ion source, or by any other interaction of constituents of the ion source with the sample and substrate.
In both embodiments of this second class of the invention, if analytes are present in solution, solvent may be removed by evaporation in the process of sample application to the requisite substrate. Both embodiments provide data in the form of isotope concentration as a function of position on the substrate. With appropriate knowledge of how samples were applied to the substrate, such data can be transformed to recreate a prior relationship between samples, as for example, the temporal or volumetric relationship between different components in the eluent of a chromatograph.
Depending on which input technology is selected, measurements are performed in one of two regimes of operation: (i) high resolution, or (ii) high throughput. Coupling the AMS system to a liquid chromatograph, capillary electrophoresis (CE), or other liquid-phase sample separation technique results in high-resolution measurements. Reconfiguration of the interface to couple small volume, microfluidic devices, or to introduce samples by laser induced desorption results in a high throughput analytical system. The second class of embodiments wherein sample is applied to a solid substrate have the advantage that they can be used both in the high resolution and in the high throughput mode.
Several embodiments of the present invention that are examples of these different regimes of operation include: (1) coupling of an LC to an AMS system with desolvation, for the analysis of 14C-labeled samples, (2) coupling of an LC to an AMS system without desolvation, for the analysis of 3H-labeled samples, (3) coupling a microfluidic device to the AMS system, (4) using laser induced desorption as the initial step in preparing samples for analysis by AMS, and (5) using laser-induced conversion to analyze either an LC chromatogram or a discrete (unfractionated) sample. Although the specific examples presented involve the detection of 14C or 3H, the technique applies to the detection of any low abundance isotope detectable by AMS, or the detection of other isotopes by other analytical techniques, such as IRMS.
These and other objects, features and advantages of the present invention will become apparent in light of the following detailed description of preferred embodiments thereof, as illustrated in the accompanying drawings.